Researchers have developed an optical system that enables the use of a single laser for multi-color STED microscopy. The cost and complexity of STED microscopes will be greatly reduced with the configuration developed by Prof. Ha that takes the light of a laser source, divide it, filter it and use it for STED imaging. The system has already been tested for dual color imaging and the use of dynamic filters will enable the simple introduction of more colors.

This new pump-probe fluorescence microscope provides efficient, high-resolution, lifetime imaging of ultrafast fluorescence phenomena. It provides an economical alternative to existing confocal and fluorescence microscopes. This microscope provides superior resolution to standard fluorescence and two-photon microscopes and comparable resolution to confocal microscopes. In addition, it eliminates the problem of precision alignment that must be maintained with confocal microscopes.

Fluorescence lifetime imaging allows researchers to see specimen changes in response to various stimuli. While the intensity of an induced fluorescent signal has long been the primary parameter for characterizing sample structures, dynamic changes in samples over time also provide valuable information. Dynamic processes can be examined by monitoring the fluorescence induced in response to sinusoidally modulated excitation lasers. Imaging of cells throughout the lifetime of a typical chromophore is necessary to obtain useful information concerning phase and modulation; however, that lifetime is on the order of one nanosecond.

This pump-probe stimulated emission microscope obtains these phase and modulation data via two modulated lasers operating at two different frequencies. The two lasers project beams upon an autofluorescent or stained sample in a spatially overlapped fashion. The first laser (pump laser) is focused onto a diffraction-limited spot to excite a fluorescent sample, while the second laser (probe laser) is focused on the same spot to induce a stimulated fluorescent emission. Optics (e.g., scanning mirrors) combine and focus the beams to perform a raster scan, in accordance with standard video-formatting techniques.

A cross-correlation signal, generated by sample fluorescence, produces the beneficial axial sectioning and is strongly dependent upon efficient spatial overlapping of the pump and probe beams at the focal point. Spatial resolution is obtained by detecting the fluorescence at the beam frequency or its harmonics. The detector that senses the responsive fluorescence need only have a temporal resolution to sense the low-frequency, cross-correlation signal.

Output of the sensor is digitized and sampled, and its Fourier transform is taken. Image data are obtained from either the phase or modulation (or both) of the resulting signal (i.e., the signal that has been sampled and transformed). By varying the polarization of the pump beam relative to the probe beam, this design also provides a way to measure polarization relaxation of excited state molecules. Design variations also allow for three-dimensional sectioning of thick samples.

Why It Is Better

Existing fluorescence, confocal, and two-photon microscopes that typically are used in this type of research have limitations, and this pump-probe stimulated emission microscope overcomes these limitations. Standard fluorescence microscopes offer poor spatial resolution because of off-focal fluorescence interference. They also cannot provide three-dimensional section imaging. Confocal microscopes use a spatial filter in front of the optical sensor to prevent off-focal fluorescence from reaching the optical sensor. They offer much better spatial resolution than standard fluorescence microscopes and also allow for three-dimensional section imaging. The drawback is that confocal microscopes are expensive and require very precise mechanical alignment that can be difficult to maintain. Two-photon microscopes prevent off-focal fluorescence, provide three-dimensional section imaging, and alleviate the problems of alignment; however, they are very expensive.

Another problem with existing fluorescence microscopy is limitation of the temporal resolution of the optical detectors to the maximum frequency of the fluorescence phenomena from which information concerning individual fluorescence lifetimes is gathered. These microscopes fail to take advantage of the speed of the excitation laser systems that operate at frequencies up to 220 GHz.

This new microscope design eliminates the off-focal fluorescence problem and provides superior spatial resolution by detecting the fluorescence at the beat frequency or its harmonics. Compared with confocal microscopes that are expensive and require very precise mechanical alignment, this new microscope design is compact, costs less, and does not require precision maintenance of the alignment.

Furthermore, to overcome the speed limitations of conventional photomultipliers, this design uses cross-correlation frequencies that range up to the 10-kHz limit of the photomultiplier, a property which prevents the imposition of limits on the temporal resolution of the overall system and allows for imaging of ultrafast fluorescence phenomena.

A near-field microscope using one or more diffractive elements placed in the near-field of an object to be imaged. A diffractive covers the entire object, thus signal may thereby be gathered from the entire object, and advantageously increase the signal-to-noise ratio of the resulting image, as well as greatly improve the acquisition speed. Near-field microscopy overcomes the limitation of conventional microscopy in that subwavelength and nanometer-scale features can be imaged and measured without contact.

Conventional Near-field Scanning Optical Microscopes (NSOM) and Atomic Force Microscopes (AFM) used for collecting images of sub-wavelength features in nanostructures and biological samples are inherently slow and can damage the object they are imaging. Their resolution is limited by the aperture of the scanning tip; hence a low signal-to-noise ratio is achieved. This Near-field Diffractive Microscope (NDM) addresses these issues by collecting data from the entire scattering surface simultaneously without the need for a small aperture to achieve high resolution images. The Near-field Diffractive Microscope uses a Fresnel plate as a single diffractive element to provide all the spatial frequencies needed to collect exhaustive information from different regions of the sample.

This optical device provides several benefits over the existing instruments, including increased speed of data collection and enhanced resolution. It has the potential to be of use in the imaging of samples from various fields - from life sciences to material science and in microlithography - where the ability to resolve atomic scale features with great speed and accuracy are crucial.

Interferometric Synthetic Aperture Microscopy (ISAM) ISAM offers revolutionary technology advancement in OCT and other microscopy methods. With a single pass, the algorithm is able to extend the range over which devices can scan an image by integrating data taken from non-focal point areas in addition to the focal region. Integration of the ISAM technology into catheters or arterial imaging devices would add significant value above the existing image rendering paradigms.

Benefits

Produces functional imagery from formerly unusable data

Maintains quality resolution in high depth-of-field and 3-D applications

Tolerates errors in defocus

Employs digital processing to compensate for instrument and user error

Methods and apparatus for testing a microscale or nanoscale sample. A testing stage comprises a frame having first and second laterally opposing ends and first and second side beams. At least one deformable force sensor beam is disposed near the first opposing end and extends laterally between the first and second side beams.

A first longitudinal beam, having a free end, bisects the at least one force sensor beam, and a second longitudinal beam has a free end facing the free end of the first longitudinal beam to define a gap therebetween. A support structure comprises a plurality of laterally extending beams disposed such that the second longitudinal beam bisects the plurality of laterally extending beams.

Each of a pair of slots disposed at each of the free ends of the first and second longitudinal beams comprises a tapered portion leading to a generally longitudinal portion aligned with the central longitudinal beam. The slots provide a seat for a dogbone-shaped sample.

This device allows for the analysis of mechanical responses of micro scale samples through uniaxial-tensile and compressive testing. In order to be tested, the externally created sample simply has to be shaped like a dog bone.

The location of the measurement gauge permits the viewer to examine the sample and the distortion value simultaneously. This allows the viewer to better observe the microstructural changes during loading and unloading.

The device also reduces steps and errors through its nano-indentation calibration system. It requires only one simple and external calibrator. This limits feasible errors in measuring the deflection of the leaf springs to less than 0.02%.

This technology can be primarily applied to mechanical testing of micro-scale materials.

Interferometric Synthetic Aperture Microscopy (ISAM) is a method of tomographic optical microscopy that brings the power of computed imaging and inverse scattering together with interferometric broadband optical imaging. ISAM provides an extended three-dimensional resolution of objects, including regions away from the focus of the objective, and quantitative estimation of the inhomogeneities in refractive index or susceptibility of an object. Using a scanned beam in regular ISAM may have disadvantages, because, for very fast phenomena, the beam may not scan fast enough. While fully coherent ISAM provides the ability to take pictures in three-dimensional volumes in a fraction of a section, it is difficult to use in highly scattering samples. By decreasing the coherence one can reduce the multiple scattering component; however, the solution for the partially coherent ISAM case is different and therefore needs a separate derivation to find the mathematical relationship between object structure and data.

This invention is full-field optical coherence tomographic microscopy with a source of varying spatial coherence.

Description/Details

The illumination system consists of a spatially incoherent source (e.g. filament of an incandescent light bulb), an iris to vary the apparent size of the source, and a collimation lens to collimate the source illumination. This system produces an adjustable partially coherent illumination at the source field plane.

Data is acquired by tuning the wavelength of the source over a particular bandwidth while recording the intensity of interferograms on the focal plane array. A linear solution for the inverse scattering problem for partially coherent illumination is derived by computational models.

Applications

Tumor analysis

Evaluation of surface topography

Fluorescent microscopy

Ideal for bench-top microscope-based systems

Benefits

Extended three-dimensional resolution

Adjustable partially coherent illumination

Fixes sample scatter and optics issues

Less expensive as it does not require a separate reference telescope to relay the reference field

It is currently difficult to determine the shapes of nanoparticles. Current methods rely on fluorescence or Raman-spectroscopy, yet these methods are not always reliable, especially in high-noise environments, and they do not offer a three-dimensional characterization of the nanoparticle. This invention is a technique to identify shapes inferred from scattering properties of nanoparticles with no prior knowledge of the shapes.

This technique includes a modification of a coherent confocal microscope device, as well as an algorithm to analyze the microscopes observations. A coherent confocal microscope device is equipped with a beam shaper that controls polarity of the incident beam. Interference with a reference beam allows the collection of data sensitive to an electric field. A high aperture lens gives many propagation directions and therefore many polarization states in the field. The field in the focal region is found by integrating the incident rays in an angular spectrum. The resulting focal fields display significant fields in all directions. The scattered field can then be propagated back to the detector.

Diverse PSFs/OTFs mean each component of the polarizability produces a different signature in the data. Assuming a single isolated scatterer, the polarizability and position can be estimated by minimizing a cost function. Near the focal plane, each OTF can be approximately characterized by one magnitude and one phase function. The approximation makes it easy to repeatedly calculate the cost. A Nelder-Mead algorithm is used to iteratively minimize over the three position variables.

Applications

This invention can determine shapes at the virus level and distinguish between different viruses for screening purposes

The Near-field Volume Scanning Optical Tomography (NVSOT) improves upon the existing Near-field Scanning Optical Microscopy (NSOM) modalities by extending the data acquisition to obtain the tomographic information of the sample without multi-directional measurements or multi-angle illuminations. Three-dimensional imaging is a much desired capability in the field of life sciences and nanotechnology; current techniques require multiple image acquisitions and complicated optical set-up.

Measurement of the volume above the sample with a strongly scattering tip in the NVSOT based system induces higher order interactions which contains the tomographic information required to generate a three-dimensional image of the sample.

Near-field Volume Scanning Optical Tomography (NVSOT) enables collection of tomographic data for the reconstruction of three dimensional images of sub-wavelength scale samples in the near-field of a strongly scattering tip. This technology eliminates the need for multi-directional measurements of the scattered field in the illumination mode or multi-angle illuminations in the collection mode. Scanning the three dimensional volume above the sample amounts to acquiring multiple NSOM images, data independence is ensured by the higher order interactions between the probe tip and the sample.

Applications

For optical microscopy and three-dimensional imaging of sub-wavelength scale samples in the fields of:

Biological Sciences

Material Sciences

Nanotechnology

Benefits

Volume scan of the surface above the sample with a strongly scattering tip enables tomographic reconstruction of the sample.

Eliminates the need for multi-directional measurements of the scattered field in the illumination mode.

Eliminates the need for multi-angle illumination for measurements in the collection mode.

The higher order interaction between the sample and the tip ensure data independence.

The development of near-field scanning optical microscopy (NSOT) has been driven by the need for an imaging technique that retains the various contrast mechanisms afforded by optical microscopy methods while attaining spatial resolution beyond the classical optical diffraction limit.

NSOT, as an extension of near-field scanning optical microscopy (NSOM), amounts to inverting the scattering data collected by the NSOM and reconstructing the sample, instead of taking the raw data as an image of the sample. This invention is a new method to simplify realization of NSOT and reduce mechanical and other noises. This invention also proposes an alternative NSOT modality to improve the experimental feasibility of NSOT.

For ultra-high optical resolution, near-field scanning optical microscopy (NSOM) is currently the photonic instrument of choice. Near-field imaging occurs when a sub-micron optical probe is positioned a very short distance from the sample and light is transmitted through a small aperture at the tip of this probe. The near-field is defined as the region above a surface with dimensions less than a single wavelength of the light incident on the surface. Within the near-field region evanescent light is not diffraction limited and nanometer spatial resolution is possible. This phenomenon enables non-diffraction limited imaging and spectroscopy of a sample that is simply not possible with conventional optical imaging techniques.

In addition, rather than using multiple observation angles or multiple probes, it is possible to collect data in a third dimension using the spectral degree of freedom. The idea of constructing an image in N spatial dimensions by collecting data in (N - 1) spatial dimensions and a spectral dimension has found application in techniques such as optical coherence tomography and synthetic aperture radar.

Applications

Any high magnification microscopy, with requirements short of a scanning electron microscope.

Benefits

This method clarifies the ambiguitybetween the sample and its NSOM image, and by inverting multiple NSOM images of a three-dimensional sample, a three-dimensional tomography of the sample may be obtained.

Multiple images corresponding to different wavelengths can be collected from each probe scan and are therefore inherently co-registered.

The development of near-field scanning optical microscopy (NSOT) has been driven by the need for an imaging technique that retains the various contrast mechanisms afforded by optical microscopy methods while attaining spatial resolution beyond the classical optical diffraction limit.

NSOT, as an extension of near-field scanning optical microscopy (NSOM), amounts to inverting the scattering data collected by the NSOM and reconstructing the sample, instead of taking the raw data as an image of the sample. This invention is a new method to simplify realization of NSOT and reduce mechanical and other noises. This invention also proposes an alternative NSOT modality to improve the experimental feasibility of NSOT.

For ultra-high optical resolution, near-field scanning optical microscopy (NSOM) is currently the photonic instrument of choice. Near-field imaging occurs when a sub-micron optical probe is positioned a very short distance from the sample and light is transmitted through a small aperture at the tip of this probe. The near-field is defined as the region above a surface with dimensions less than a single wavelength of the light incident on the surface. Within the near-field region evanescent light is not diffraction limited and nanometer spatial resolution is possible. This phenomenon enables non-diffraction limited imaging and spectroscopy of a sample that is simply not possible with conventional optical imaging techniques.

In addition, rather than using multiple observation angles or multiple probes, it is possible to collect data in a third dimension using the spectral degree of freedom. The idea of constructing an image in N spatial dimensions by collecting data in (N - 1) spatial dimensions and a spectral dimension has found application in techniques such as optical coherence tomography and synthetic aperture radar.

Applications

Any high magnification microscopy, with requirements short of a scanning electron microscope.

Benefits

This method clarifies the ambiguity between the sample and its NSOM image, and by inverting multiple NSOM images of a three-dimensional sample, a three-dimensional tomography of the sample may be obtained.

Multiple images corresponding to different wavelengths can be collected from each probe scan and are therefore inherently co-registered.